Anti-microbial seaweed extracts, compositions and uses thereof

ABSTRACT

The present invention provides a crude extract, fractions and sub-fractions from the seaweed Fucus distichus (FD), method of preparation and its use for inhibiting the growth of microbial cells, particularly bacteria causing acne or nosocomial infections such as MRSA in humans or MRSP in dogs.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. Ser. No. 16/398,779,filed Apr. 30, 2019 as a divisional of U.S. Ser. No. 15/528,372, filedMay 19, 2017, which is the U.S. National Stage of International PatentApplication No. PCT/CA2015/051310, filed Dec. 11, 2015, each of which ishereby incorporated by reference in its entirety, and which claimpriority to U.S. Provisional Patent Application No. 62/090,973, filedDec. 12, 2014.

FIELD OF THE INVENTION

The present invention relates to extracts from the seaweed Fucusdistichus (FD), method of preparation and use for inhibiting the growthof microorganisms such as bacteria

BACKGROUND OF THE INVENTION

Acne vulgaris is a common cutaneous multifactorial disease spreadworldwide and caused by hormonal, microbiological and immunologicalmechanisms. Acne is characterized by open and closed comedones(blackheads and whiteheads) and inflammatory lesions like papules,pustules and nodules. Staphylococcus aureus, Staphylococcus epidermidisand Propionibacterium acnes are the organisms which proliferate rapidlyand cause development of acne. The severity of this skin disordergenerally increases with age and time. People normally get affected byit with the onset of puberty affecting both physical & psychologicallevels and therefore may constitute a cause of concern for treatingphysicians.

Acne affects all age groups i.e. 85% of teenagers, about 8% in 25-34year olds and 3% in 35-44 year olds. Although it is not a lifethreatening disease, it is a distressing skin condition which causessignificant psychological disability. Moreover, teenagers or youngadults often experience the development of scar and scarring may affectup to 95% of the patients having acne.

There is a large and expanding market for over-the-counter (OTC)medications against acne. The estimated annual worldwide expenditure onacne OTC medication is $100 millions. The long term treatment of thepresent synthetic drugs comprising antibiotics and chemotherapeuticagents either inhibit excess sebum production, follicularhyperkeratinisation disorders, cytokines, reactive oxygen species andproliferation of P. acnes within the follicle. These drugs are appliedeither topically or taken orally for the treatment of acne.

The therapeutic success in the treatment of acne is highly dependent onthe regular application of topical agents over a prolonged period oftime. However the disadvantages associated with the existing topicaltherapies defeat the purpose of the treatment and make itpatient-noncompliant. Currently available treatment for acne is based onantibiotics and retinoids. The use of antibiotics have lot oflimitations due to the development of resistance by bacteria and theiruntoward side effects, such as skin dryness, pruritis, burningsensation, erythema, occasional hyper pigmentation, local irritation andphotosensitization reactions. Furthermore, retinoids are highlyteratogenic.

Also, extracts from plants and specific compounds obtained from plantsources are often used in cosmetic and pharmaceutical compositions.European Patent Application Publication No. 0 870 507 describes asynergistic antibacterial composition that includes an extract ofbotanical materials and an essential oil. The essential oil is describedas having anti-microbial activity, whereas the extract of botanicalmaterials has significantly lower activity, or no anti-microbialactivity, when used alone.

Therefore alternative treatments of acne using natural products must bestudied and developed. This creates a great interest in development of atopical formulation containing natural extracts possessing antibacterialeffect to treat acne such as the development of present invention.

Nosocomial infections are hospital-acquired infections (HAI) orhealthcare-acquired infections whose development is favored by ahospital environment, such as one acquired by a patient during ahospital visit or one developing among hospital staff. In the UnitedStates, the Centers for Disease Control and Prevention estimated roughly1.7 million hospital-associated infections, from all types ofmicroorganisms (i.e. bacteria), combined, cause or contribute to 99,000deaths each year. Nosocomial infections can cause severe pneumonia andinfections of the urinary tract, bloodstream and other parts of thebody. Many types are difficult to treat with antibiotics, and antibioticresistance is spreading to Gram-negative bacteria that can infect peopleoutside the hospital.

Methicillin-resistant Staphylococcus aureus (MRSA) is responsible forseveral difficult-to-treat infections in humans. MRSA is any strain ofStaphylococcus aureus that has developed, through resistance tobeta-lactam antibiotics, such as the penicillin-types (methicillin,dicloxacillin, oxacillin, etc.) and the cephalosporins. This resistancemakes MRSA infection more difficult to treat with standard types ofantibiotics and thus more dangerous.

MRSA is especially troublesome in hospitals, prisons, and nursing homes,where patients with open wounds, invasive devices, and weakened immunesystems are at greater risk of nosocomial infection than the generalpublic. MRSA began as a hospital-acquired infection, but has developedlimited endemic status and is now sometimes community-acquired.

Dogs can be carriers of MRSA and may be otherwise perfectly healthy.This is referred to as colonization. Though dogs are not normallycolonized with MRSA, they can be exposed to a person that is colonizedor who has an active infection, and therefore can become infected orcolonized as well.

Staphylococcus intermedius is a common species of bacteria found inrabbits and is called Staphylococcus pseudintermedius when found indogs. A small percentage of animals may develop skin infections causedby methicillin-resistant-Staphylococcus intermedius (MRSI) or-Staphylococcus pseudintermedius (MRSP), both infections being difficultto get rid of, and often require aggressive topical therapies.

SUMMARY OF THE INVENTION

A main aspect intended to be addressed by the present invention is toprovide a novel extract from the seaweed Fucus distichus (FD).

According to a further aspect, the present invention provides acomposition comprising the extract as defined herein, in admixture witha physiologically acceptable excipient.

According to a further aspect, the present invention provides a methodfor inhibiting a microorganism comprising contacting said cell with agrowth-inhibiting concentration of the extract or the composition asdefined herein.

According to a further aspect, the present invention provides a methodfor treating a microbial infection in a mammal comprising administeringa growth-inhibiting concentration of the extract of the composition asdefined herein to the mammal.

According to a further aspect of the present invention, there isprovided use of the extract as defined herein for inhibiting growth ofmicrobial cells.

According to a further aspect of the present invention, there isprovided use of the extract as defined herein for the manufacture ofcomposition for treating a microbial infection in a mammal.

According to a further aspect, the present invention provides use of thecomposition as defined herein for the treatment of a microbial infectionin a mammal. Particularly, the microbial infection is a bacterialinfection or an antibiotic-resistant bacterial infection.

According to a further aspect of the use or the method, both as definedabove, the bacterial infection may be selected from the group consistingof: Staphylococcus aureus, methicillin-resistant Staphylococcus aureus(MRSA), Staphylococcus epidermidis, Propionibacterium acnes,Staphylococcus intermedius (SI), methicillin-resistant Staphylococcusintermedius (MRSI), Staphylococcus pseudintermedius (SP) andmethicillin-resistant Staphylococcus pseudintermedius (MRSP).

According to a further aspect, the present invention provides a methodfor obtaining an extract from Fucus distichus comprising the steps of:a) mixing material from seaweed Fucus distichus (FD) with a solvent toobtain a solvent:material mixture; and b) separating a solid fractionand a liquid fraction from said mixture, said liquid fraction formingthe extract.

DETAILED DESCRIPTION OF THE INVENTION Description of the Figures

FIG. 1. Photograph of Fucus distichus seaweed.

FIG. 2. Evaluation by luminescence of anti-microbial activity of aprimary extract of FD against methycillin-resistant Staphylococcusaureus (MRSA).

FIG. 3. Evaluation by luminescence of anti-microbial activity of aprimary extract of FD against Staphylococcus epidermidis.

FIG. 4. Evaluation by luminescence of anti-microbial activity of aprimary extract of FD against Proprionibacterium acnes.

FIG. 5. Fractionation strategy for secondary fractionation of primary FDextracts.

FIG. 6. Turbidimetry of Staphylococcus pseudintermedius according totime-points; bacterial suspension 10³ CFU-mL.

FIG. 7. Turbidimetry of Staphylococcus pseudintermedius; bacterialsuspension 10³ CFU-mL in Mueller Hinton medium.

FIG. 8. Anti-microbial activity of a primary extracts of FD againstStaphylococcus pseudintermedius.

Abbreviations and Definitions Abbreviations

FD: Fucus distichus seaweed.

Definitions

The term “about” as used herein refers to a margin of +or −10% of thenumber indicated. For sake of precision, the term about when used inconjunction with, for example: 90% means 90% +/−1-9% i.e. from 81% to99%. More precisely, the term about refer to +or −5% of the numberindicated, where for example: 90% means 90% +/−1-4.5% i.e. from 86.5% to94.5%.

As used herein the singular forms “a”, “and”, and “the” include pluralreferents unless the context clearly dictates otherwise. Thus, forexample, reference to “a cell” includes a plurality of such cells andreference to “the culture” includes reference to one or more culturesand equivalents thereof known to those skilled in the art, and so forth.All technical and scientific terms used herein have the same meaning ascommonly understood to one of ordinary skill in the art to which thisinvention belongs unless clearly indicated otherwise.

As used in this specification and claim(s), the words “comprising” (andany form of comprising, such as “comprise” and “comprises”), “having”(and any form of having, such as “have” and “has”), “including” (and anyform of including, such as “includes” and “include”) or “containing”(and any form of containing, such as “contains” and “contain”) areinclusive or open-ended and do not exclude additional, un-recitedelements or method steps.

As used herein, the terms “disease” and “disorder” may be usedinterchangeably or may be different in that the particular malady orcondition may not have a known causative agent (so that etiology has notyet been worked out) and it is therefore not yet recognized as a diseasebut only as an undesirable condition or syndrome, wherein a more or lessspecific set of symptoms have been identified by clinicians.

“Mammal” includes humans, domestic animals such as farm animals (e.g.swine, cattle, sheep, goats, horses, rabbits), household pets (e.g.cats, dogs, rabbits, hamsters, ferrets), and non-domestic animals suchas wildlife and the like.

The term “subject” or “patient” as used herein refers to an animal,preferably a mammal, and most preferably a human who is the recipient ofthe treatment, observation or experiment.

The term “extract” as used herein means a composition prepared bycontacting solvent with seaweed material, produced following theprocedures of the invention, which demonstrates inhibitory activityagainst one or more cancer cell line in vitro. In one aspect of theinvention, an extract demonstrates inhibitory activity against cancercell growth in vivo. As used herein, the term “extract” means an extractthat is: crude, fractionated, sub-fractionated, separated, isolated,enriched or purified without being limited thereto.

The term “isolated” is used herein to indicate that the protein existsin a physical milieu distinct from that in which it occurs in nature.For example, the isolated molecule may be substantially isolated (forexample enriched or purified) with respect to the complex cellularmilieu in which it naturally occurs, such as in a crude/primary extractor secondary fractions. When the isolated molecule is enriched orpurified, the absolute level of purity is not critical and those skilledin the art can readily determine appropriate levels of purity accordingto the use to which the material is to be put. In some circumstances,the isolated molecule forms part of a composition (for example a more orless crude extract containing many other substances) or buffer system,which may for example contain other components. In other circumstances,the isolated molecule may be purified to essential homogeneity, forexample as determined spectrophotometrically, by NMR or bychromatography (for example LC-MS).

The term “primary” or “crude” means compounds or molecules that have notbeen entirely separated from the components of the original compositionin which it was present. Therefore, the terms “separating”, “purifying”or “isolating” refers to methods by which one or more components of thebiological sample are removed from one or more other components of thesample.

The extracts described herein can be formulated as compositions byformulation with additives such as physiologically-acceptableexcipients, physiologically-acceptable carriers, andphysiologically-acceptable vehicles, or as cosmetic formulations withadditives such as pharmaceutically- and/or dermatologically-acceptableexcipients, carriers, and/or vehicles.

As used herein, the term “pharmaceutically-acceptable” refers beinguseful in preparing a pharmaceutical composition that is generally safe,non-toxic and neither biologically nor otherwise undesirable andincludes being useful for veterinary use as well as human pharmaceuticaluse.

As used herein, the term “dermatologically-acceptable” refers tomolecular entities and compositions that are physiologically tolerablewhen applied topically on the skin and do not typically produce anallergic or similar unwanted reaction, such as redness or swelling andthe like, when administered to human. Preferably, as used herein, theterm “cosmetically acceptable” means approved by regulatory agency ofthe federal or state government or listed in the U.S. Pharmacopeia orother generally recognized pharmacopeia for use in animals, and moreparticularly in humans.

The term “carrier” refers to a diluent, adjuvant, excipient, or vehiclewith which the compounds of the present invention may be administered.Sterile water or aqueous saline solutions and aqueous dextrose andglycerol solutions may be employed as carrier, particularly for topicalformulations. Suitable cosmetically carriers are described in“Remington's Pharmaceutical Sciences” by E. W. Martin.

DETAILED DESCRIPTION OF PARTICULAR ASPECTS OF THE INVENTION Solventextracts

With the aim of providing an alternative source of anti-microbialmolecules, there is provided a crude solvent extract from the seaweedFucus distichus (FD). Particularly, the crude extract is an organic orinorganic solvent extract. More particularly, the extract's solvent iswater or alcohol; and even more particularly: aqueous ethanol.

Particularly, the crude extract is an 80% aqueous ethanol extract of FD.More particularly, the crude extract is a previously hexane-defattedextract.

More particularly, the extract is a solvent fraction of the primaryextract. Most particularly, the fraction is obtained by a secondextraction with a solvent such as: hexane, ethyl acetate, chloroform orwater.

Extract Form

In accordance with a particular aspect of the present invention, theextract is in dried form or in solution.

Composition and/or Formulation

In accordance with a particular aspect of the invention, there isprovided a composition comprising the FD extract as defined herein, inadmixture with a physiologically- (i.e. pharmaceutically ordermatologically) acceptable carrier.

Thus, aspects of the present disclosure provide for a composition fortopical treatment of skin disorders (including acne vulgaris), thecomposition comprising an anti-microbial agent comprising the FD extractas defined herein, optionally in admixture with: one or more synergisticagent selected from the group of: anti-acne actives, anti-microbialactives, anti-fungal actives, anti-inflammatory actives, exfoliatingagents and mixtures thereof; and a physiologically-acceptable carrier.In one embodiment, the anti-microbial agent comprises the FD extract asdefined herein effective for inhibiting p. acne in aphysiologically-acceptable carrier. By way of example, the compositionmay comprise between 0.001% and 50% (w/w) active ingredients, and 50% to99.999% (w/w) physiologically-acceptable carrier.

The compositions of the invention include those suitable for oral,nasal, mucosal, rectal, topical, buccal (e.g., sub-lingual), mucosal,intraperitoneal, parenteral (e.g., subcutaneous, intramuscular,intradermal, or intravenous), topical (i.e., both skin and mucosalsurfaces, including airway surfaces) and transdermal administration,although the most suitable route in any given case will depend on thenature and severity of the condition being treated and on the nature ofthe particular active compound that is being used.

Compositions suitable for oral administration may be presented indiscrete units, such as capsules, cachets, lozenges, or tablets, eachcontaining a predetermined amount of the active compound; as a powder orgranules; as a solution or a suspension in an aqueous or non-aqueousliquid or paste (such as gel, lotion, cream, ointment, etc.); or as anoil-in-water or water-in-oil emulsion. Such compositions may be preparedby any suitable method of pharmacy, which includes the step of bringinginto association the active compound and a suitable carrier (which maycontain one or more accessory ingredients as noted above).

Compositions suitable for buccal (sub-lingual) administration includelozenges comprising the active compound in a flavored base, usuallysucrose and acacia or tragacanth; and pastilles comprising the compoundin an inert base such as gelatin and glycerin or sucrose and acacia.

Compositions of the present invention suitable for parenteraladministration comprise sterile aqueous and non-aqueous injectionsolutions of the active compound, which preparations are preferablyisotonic with the blood of the intended recipient. These preparationsmay contain anti-oxidants, buffers, bacteriostats and solutes thatrender the composition isotonic with the blood of the intendedrecipient. Aqueous and non-aqueous sterile suspensions may includesuspending agents and thickening agents. The compositions may bepresented in unit/dose or multi-dose containers, for example sealedampoules and vials, and may be stored in a freeze-dried (lyophilized)condition requiring only the addition of the sterile liquid carrier, forexample, saline or water-for-injection immediately prior to use.Extemporaneous injection solutions and suspensions may be prepared fromsterile powders, granules and tablets of the kind previously described.

For example, in one aspect of the present invention, there is providedan injectable, stable, sterile composition comprising an active compoundas described herein, or a salt or prodrug thereof, in a unit dosage formin a sealed container. The compound or salt is provided in the form of alyophilizate that is capable of being reconstituted with a suitablepharmaceutically acceptable carrier to form a liquid compositionsuitable for injection thereof into a subject. The unit dosage formtypically comprises from about 10 mg to about 10 grams of the compoundor salt. When the compound or salt is substantially water-insoluble, asufficient amount of emulsifying agent that is physiologicallyacceptable may be employed in sufficient quantity to emulsify thecompound or salt in an aqueous carrier. One such useful emulsifyingagent is phosphatidyl choline.

Compositions suitable for rectal administration are preferably presentedas unit dose suppositories. These may be prepared by mixing the activecompound with one or more conventional solid carriers, for example,cocoa butter, and then shaping the resulting mixture.

In an alternative embodiment, the present composition may beadministered via topical administration.

Compositions suitable for topical application to the skin preferablytake the form of an ointment, cream, salve, foam, lotion, paste, gel,spray, aerosol, or oil. Carriers that may be used include petroleumjelly, lanoline, polyethylene glycols, alcohols (e.g., ethanol,isopropanol, etc.), transdermal enhancers, and combinations of two ormore thereof.

Alternatively, the present composition may be formulated in amicrocrystalline form, in a liposomal preparation or as a wipe. Thepresent composition may be formulated to be used as a cleanser or atoner. The present composition may be formulated to be used on the wholesurface of a target skin area or for spot skin treatment. Formulationssuitable for a desired route of administration are within the skill ofone in the art.

Inactive Ingredients and Carriers

The composition of the present invention may comprise, in addition tothe active agent, one or more inactive ingredient selected from thegroup consisting of: carriers or excipients, viscosity or buildingagents, thickening agents, gelling agents and preservative agents.

The pharmaceutical compositions of the present invention can beformulated based on their routes of administration using methods wellknown in the art. For example, a sterile injectable preparation can beprepared as a sterile injectable aqueous or oleaginous suspension usingsuitable dispersing or wetting agents and suspending agents.Suppositories for rectal administration can be prepared by mixing drugswith a suitable non-irritating excipient such as cocoa butter orpolyethylene glycols which are solid at ordinary temperatures but liquidat the rectal temperature and will therefore melt in the rectum andrelease the drugs. Solid dosage forms for oral administration can becapsules, tablets, pills, powders or granules. In such solid dosageforms, the active compounds can be admixed with at least one inertdiluent such as sucrose lactose or starch. Solid dosage forms may alsocomprise other substances in addition to inert diluents, such aslubricating agents. In the case of capsules, tablets and pills, thedosage forms may also comprise buffering agents. Tablets and pills canadditionally be prepared with enteric coatings. Liquid dosage forms fororal administration can include pharmaceutically acceptable emulsions,solutions, suspensions, syrups or elixirs containing inert diluentscommonly used in the art. Liquid dosage forms may also comprise wetting,emulsifying, suspending, sweetening, flavoring, or perfuming agents. Thepharmaceutical compositions of the present invention can also beadministered in the form of liposomes, as described in U.S. Pat. No.6,703,403. Formulation of drugs that are applicable to the presentinvention is generally discussed in, for example, Hoover, John E.,REMINGTON'S PHARMACEUTICAL SCIENCES (Mack Publishing Co., Easton, Pa.:1975), and Lachman, L., eds., PHARMACEUTICAL DOSAGE FORMS (MarcelDecker, New York, N.Y., 1980).

The choice of a suitable physiologically-acceptable carrier will dependon the exact nature of the particular formulation desired, e.g. whetherthe present topical composition is to be formulated into a liquidsolution, a suspension, an ointment, a film or a gel. The choice of asuitable physiologically-acceptable carrier will also depend on theroute of administration. Preferably, the carrier is formulated to besuitable for topical administration.

In accordance with a particular embodiment, the inactive ingredient maybe: a polyacrylate, carbopol 940,934,970,974, acacia, alginic acid,bentonite, carboxymethylcellulose, ethylcellulose, gelatin,hydroxyethylcellulose, hydroxypropyl cellulose, magnesium aluminumsilicate, methylcellulose, poloxamers, polyvinyl alcohol, sodiumalginate, tragacanth, and xanthan gum or mixtures thereof.

In still another embodiment, preservatives like paraben andtriethanolamine may be added to increase the stability of thecomposition.

In the case of a topical formulation in a gel form, the carrier may beselected from the group consisting of: purified water; ammoniumacryloyldimethyltaurate; VP colopolymer; aloe vera; edetate disodium;allantoin; methylchloroisothiazolinone; methylisothiazolinone; andmixtures thereof.

Alternatively, the present composition may be formulated as ananti-bacterial soap or detergent, for preventive or hygienic purposes.Particularly, in one embodiment, the anti-microbial detergent comprisesan extract of the present invention in combination with one or moreadditional cleaning composition components. The choice of additionalcomponents is within the skill of the artisan and includes conventionalingredients, including the exemplary non-limiting components set forthherein.

The detergent composition may be suitable for washing skin or mucusmembranes (mouthwash, nose drops or rinse, etc.), or cleaning hardsurfaces such as e.g. floors, tables, or dish wash.

Use and Method of Treatment

In accordance with an alternative aspect, the present invention providesthe use of the extract as defined herein for inhibiting growth ofmicrobial cells. Particularly, there is provided the use of the extractas defined herein for the manufacture of composition for treating amicrobial infection in a mammal.

In accordance with an alternative aspect of the invention, there isprovided the use of the composition as defined herein for the treatmentof a microbial infection in a mammal.

In accordance with a particular aspect, the present invention provides amethod of inhibiting a microbial cell growth comprising contacting saidcell with a growth-inhibiting concentration of the extract as definedherein or the composition as defined herein.

More particularly, there is provided a method of treatment of amicrobial infection in a mammal comprising administering agrowth-inhibiting concentration of the composition as defined herein tosaid mammal.

In another aspect of the present disclosure, there is provided a methodfor the treatment of a skin disorder in a subject in need thereof,wherein the method comprises administering to the subject atherapeutically effective amount of a composition comprising ananti-microbial amount of the FD extract as defined herein in admixturewith a physiologically-acceptable carrier. In one embodiment, theadministering is topical, whereby the treatment is applied to a skinarea affected by the bacterial infection. Compositions suitable for thepresent method are disclosed herein.

In another aspect of the present disclosure, there is provided a use ora method for the treatment of MRSA in a pet, particularly a dog, whereinthe method comprises administering to the pet a therapeuticallyeffective amount of a composition comprising an anti-microbial amount ofthe FD extract as defined herein in admixture with apharmaceutically-acceptable carrier. In one embodiment, theadministering is topical, whereby the treatment is applied to a skinarea affected by the MRSA infection. Compositions suitable for thepresent method are disclosed herein.

In a further aspect of the present disclosure, there is provided use anda method for the treatment of Staphylococcus pseudointermedius (SP) ormethicillin-resistant SP in a pet, particularly a dog, wherein themethod comprises administering to the pet a therapeutically effectiveamount of a composition comprising an anti-microbial amount of the FDextract as defined herein in admixture with apharmaceutically-acceptable carrier. In one embodiment, theadministering is topical, whereby the treatment is applied to a skinarea affected by the SP infection. Compositions suitable for the presentuse and method are disclosed herein.

In a further aspect of the present disclosure, there is provided use anda method for the treatment of Staphylococcus intermedius (SI) ormethicillin-resistant SI in a farm animal, particularly a rabbit,wherein the method comprises administering to the rabbit atherapeutically effective amount of a composition comprising ananti-microbial amount of the FD extract as defined herein in admixturewith a pharmaceutically-acceptable carrier. In one embodiment, theadministering is topical, whereby the treatment is applied to a skinarea affected by the SI infection. Compositions suitable for the presentuse and method are disclosed herein.

Bacterial Infection

According to a further aspect, the microbial infection is a bacterialinfection or an antibiotic-resistant bacterial infection. Particularly,the bacterial infection may be selected from the group consisting of:Staphylococcus aureus, methicillin-resistant Staphylococcus aureus(MRSA), Staphylococcus epidermidis, Propionibacterium acnes,Staphylococcus intermedius (SI) and methicillin-resistant Staphylococcusintermedius (MRSI), Staphylococcus pseudintermedius (SP) andmethicillin-resistant Staphylococcus pseudintermedius (MRSP).

Subject

In accordance with another aspect, the mammal may be a human, a farmanimal or a pet such as, for example, horses, rabbits, cats or dogs,particularly dogs.

Cosmetic Indications

The present invention also provides for a use or a method foralleviating acne-associated symptoms, the method comprises administeringto a skin area affected by acne a therapeutically-effective amount of acomposition comprising the FD extract directed against the organismsassociated with acne and a physiologically-acceptable carrier,optionally in admixture with one of: anti-acne actives, anti-microbialactives, antifungal actives, anti-inflammatory actives, exfoliatingagents and mixtures thereof. Compositions for alleviatingacne-associated symptoms are disclosed herein.

Method of Extraction

In accordance with a further aspect of the invention, there is provideda method for obtaining an extract from Fucus distichus (FD) comprisingthe steps of:

-   -   a) mixing material from seaweed Fucus distichus with a solvent        to obtain a solvent: material mixture; and    -   b) separating a solid fraction and a liquid fraction from said        mixture, said liquid fraction forming said extract from said        seaweed material.

Particularly, the solvent is organic or inorganic; more particularly:water or alcohol; and most particularly: aqueous ethanol. Still, mostparticularly, the solvent is 80% aqueous ethanol.

In accordance with an alternative aspect, the method of the inventionfurther comprises a hexane-defatting step prior to step a).

In accordance with a particular aspect, the method further comprises thestep of: c) fractionating the extract from step b) with a furthersolvent selected from the group consisting of: hexane, ethyl acetate,chloroform, water and mixtures thereof to obtain a liquid fraction.

Alternatively, the method further comprises a step of drying the liquidfraction to obtain a dried extract.

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Centigrade,and pressure is at or near atmospheric.

EXAMPLES

This disclosure describes Fucus distichus harvesting, preparation ofextracts, and testing for anti-microbial activity.

Example 1 Seaweed Collection and Identification

In September 2008, Fucus distichus (FD) was collected by hand from BonneBay, Newfoundland, Canada. Samples were placed in plastic sampling bagsand transported to Applicant's premises in coolers of seawater. Uponarrival in the laboratory, the specimens were washed individually toremove epiphytic and extraneous matter (sand, mussels, isopods, etc.).Samples were then checked visually to ensure they were clean. If not,remaining matter was removed by hand with further washing. Seaweeds wereblotted dry, weighed to the nearest gram (Plant wet weight) andshredded. The shredded material was transferred into Erlenmeyer flasksand frozen at −60° C. until the extracts were prepared.

A representative sample was also photographed (FIG. 1) and frozen at−20° C. for confirmation of species by Dr. Robert Hooper, a phycologistat Memorial University of Newfoundland.

Extract Preparation

Preparation of extract involved freeze drying and de-fatting samples,followed by extraction with 20% to 80% aqueous ethanol (FIG. 5).

Freeze-Drying

Seaweeds were freeze-dried prior to extraction. This step accounts forthe differences in water content among seaweeds which may otherwiseaffect the solubility of bioactive components. Secondary plantmetabolites are also more stable when stored in a dried form. Moreover,the large scale extraction of dried plant material may cause fewerproblems than extracting fresh material. In order to preservethermo-labile compounds, low temperature conditions are used throughoutthe process of extraction.

Erlenmeyer flasks containing the shredded seaweeds, which had beenfrozen at −60° C., were placed on a freeze-dryer, and lyophilized for72-96 h at 69×10⁻³ mbar. The weight (g) of dry material was thenrecorded.

Defatting of Samples

The lipid fraction of seaweed is known to vary from 1 to 5% of the algaldry matter, which can be dominated by polyunsaturated fatty acids. Brownand red seaweeds are particularly rich in long chain polyunsaturatedfatty acids such as eicosapentaenoic acid (n3, C20:5), while greenseaweeds may possess a level of alpha linoleic acid (n3, C18:3). Sincethese polyunsaturated fatty acids are extremely susceptible tooxidation, they may result in lipid oxidation products during analysis.In order to eliminate the above oxidative processes that may have aneffect on the results, samples were defatted prior to extraction.

Freeze dried seaweed samples were ground into a powder and defatted byblending the powder with hexane (1:5, w/v, 5 min) in a Waring blender atambient temperature. Defatted samples were air-dried, vacuum packed inpolyethylene pouches and kept at 4° C. until extraction.

Example 2 Crude Extraction

Different solvents or solvent systems can be used for the extraction. Ingeneral, ethanol is commonly used due to its lower toxicity compared toother solvents. Moreover, ethanol extracts have been demonstrated inmany studies to have the highest antioxidant activity.

In the current study, bioactive compounds were extracted into 80%aqueous ethanol at 4° C. for 24 h. The solvent was then removed under avacuum at 37° C. for 45 to 60 min and the resulting concentratedslurries were lyophilized for 72 to 96h at −80° C. and 69×10⁻³ mbarusing a freeze dryer. Dry extracts were weighed and stored at −60° C.until preparation for screening.

Extraction Yield

Extraction yields were calculated and expressed as g of dry extract perkg of dry seaweed. The yield was 6.24%. Twenty five (25) mg of eachextract was sent for anti-microbial screening assays.

Example 3. Primary Anti-Microbial Screening of Fucus Distichus ExtractExperimental Conditions

The samples were tested to identify their capacity to inhibit thebacterial growth of Staphylococcus epidermidis, methicillin-resistantStaphylococcus aureus (MRSA), and Propionibacterium acnes. Table 1enumerates the characteristics of the bacterial strains and antibioticsused as positive controls while Table 2 enumerates the optimal cultureconditions.

TABLE 1 Summary of the bacterial strains characteristics, the selectedantibiotics and the catalog numbers Supplier Bacteria CharacteristicsATCC ® Antibiotics Staphylococcus Involved in nasal, 12228 Vancomycinepidermidis urinary and cutaneous infections, Gram-positiveMethicillin-resistant Involved in 43300 Chloramphenicol Staphylococcusnosocomial and aureus (MRSA) opportunistic infections, Gram-positivePropionibacterium Linked to the , 6919 Vancomycin acnes skin conditionacne, Gran-positive

TABLE 2 Summary of the experimental conditions Optimized growth BacteriaOptimal liquid medium conditions Staphylococcus Mueller Hinton Aerobic,35-37° C., epidermidis incubation 18-24 hrs Methicillin-resistantMueller Hinton Aerobic, 35-37° C., Staphylococcus incubation 18-24 hrsaureus (MRSA) Propionibacterium Brain Hearth Infusion Aerobic, 35-37°C., acnes (BHI) + vit. K + Hémine incubation 18-24 hrs

The sample (Table 3) and its solvent were tested simultaneously withantibiotic known to induce a strong inhibition of bacterial growth. Anantibiotic is used to inhibit the growth of the bacterial strains. Theseantibiotics are used as positive controls identified by “INH” in thegraphs. In addition, the extracts were tested in parallel with negativecontrols: the extracts solvent, labeled “Vehicle” and culture medium,labeled “Basal”, respectively in the graphs. To ensure thereproducibility of the biological response, all the experimentalconditions were tested in triplicate in 2 independent assays,represented by N=1 and N=2. The detection of the growth inhibition isnoted by the turbidity measurement (measurement of absorbance at 625 nm)and by the metabolic activity (luminescent determination of ATP)expressed in “Relative Luminescence Unit” (RLU). With each test, an ATPstandard curve is generated, by using the culture medium as solvent, inorder to know the quantity of ATP measured in basal condition and usingwater as solvent in order to control the inter-assay detectionvariation.

TABLE 3 Summary of the characteristics of the extract #11 ConcentrationFinal concentration with Samples Solvent (mg/ml) bacteria (mg/ml)Extract #11 DMSO 10% 25 mg/0.5 ml 50; 20; 8; 3.2 μg/ml

A growth inhibition between 50 and 74% compared to the condition withoutextract, determined by vehicle (DMSO), is regarded as a significantresponse. While a growth inhibition superior to 75% is regarded as ahighly significant answer. The comparison for the calculation of Z′ isdone between the condition of the inhibitor or the sample compared tothe vehicle. When the Z′ value approaches 1, the test is regarded asstatistically significant. When the inhibition percentage is higher than75% and the Z′ value is nearly 1 the extract is considered veryinteresting.

The observation of a dose-dependent effect of an extract can also beinteresting. Dose-dependent effect means that the effect changesproportionally when the dose of the extract is changed. An inhibitionpercentage between 50 and 74% for an extract in more than one strain canalso be very interesting. For example, extract #11 (Fucus distichus, FD)is interesting because it has an effect on three different strains:methicillin-resistant Staphylococcus aureus (FIG. 2), Staphylococcusepidermidis (FIG. 3) and Propionibacterium acnes (FIG. 4).

The results from Table 4 (below) show that FD Extract #11 hasinteresting biological activity by inhibiting growth ofmethicillin-resistant Staphylococcus aureus (MRSA), Staphylococcusepidermidis and Propionibacterium acnes.

TABLE 4 Summary of anti-microbial activity of FD extract #11 %inhibition Z′ Extracts Strains 50 μg/ml 20 μg/ml 8 μg/ml 3.2 μg/ml 50μg/ml 20 μg/ml 8 μg/ml 3.2 μg/ml 11 N = 1 S. 49 18 12 NO 0.27 N/A N = 2epidermidis 60 53 26 12 0.61 0.28 N/A 11 N = 1 SARM 62 42 25 10 0.660.16 N/A N = 2 67 34 30 10 0.60 0.37 0.25 N/A 11 N = 1 P. acnes 36 15 NO11 0.69 N/A N = 2 58 44 42 36 0.73 0.60 0.65 0.29 Legend: NO: Noinhibition, Z′ value: Statistical value.

It was therefore thought appropriate to carry out a bio-guidedfractionation of a second FD extract (#47) in order to bettercharacterize the bioactive molecules contained therein.

Example 5. Bio-Guided Fractionation of Fucus Distichus Extract #47 andEvaluation of Antimicrobial Activity

In this example, FD primary extract #47 (obtained by the same protocolas extract #11) underwent secondary fractionation by four differentsolvents. The antibacterial activity of all secondary fractions therebygenerated were tested on Propionibacterium acnes, Staphylococcusepidermidis and methicillin-resistant Staphylococcus aureus (MRSA).

Fractionation and Screening

The secondary fractionation (liquid-liquid) of FD Extract #47 wasperformed according to the solvent in which the dry extract was bestdissolved. After some preliminary tests, FD Extract #47 was found to bevery soluble in H₂O. Secondary fractionation was therefore undertaken onan aqueous solution of FD Extract #47, using three solvents of differentpolarity:

a) hexane (H), b) ethyl acetate (Ac), and c) chloroform (CL). Theremaining molecules in the solubilisation solvent (H₂O) are recovered toconstitute the fourth secondary fraction.

The screening results obtained for the secondary fractions of FD Extract#47 showed anti-microbial activity of the ethyl acetate fraction againstMRSA (Table 5), and anti-microbial activity of the chloroform fractionagainst S. epidermis and p. acnes. These results demonstrate theeffectiveness of the methodology of the bio-guided fractionation, whichconcentrates the active molecules and targets the biological effect.Moreover, we can conclude that at least some of the active moleculescontained in FD Extract #47 are polar in nature.

TABLE 5 Summary of anti-microbial activity of primary FD extract #47 andsecondary fractions MRSA S. epi 50 μg/ml 20 μg/ml 8 μg/ml 3.5 μg/ml 50μg/ml 20 μg/ml 8 μg/ml Screening Extract % Z′ % Z′ % Z′ % Z′ % Z′ % Z′ %Z′ Primary 47 63 0.67 45 0.35 28 0.45 6 NA 38 NA 4 NA 6 NA N = 1 N = 270 0.75 26 0.23 20 NA 12  NA 30 NA 48 0.5 11 NA Secondary 47H11571 260.06 17 NA 10 NA NO 2.45 10 NA 6 NA 3 NA N = 1 N = 2 13 NA 18 NA 2 NA 0NA 23 0.3 17 0.3 9 NA 47AC11571 55 0.92 14 NA 6 NA NO 1.8  34 0.3 14 NA9 NA N = 1 N = 2 49 0.57 10 NA 4 NA 5 NA 40 0.1 8 NA 1 NA 47CL11571 280.38 16 0.48 11 NA NO 2.38 30 0.2 59 0.7 26 NA N = 1 N = 2 30 0.11 230.32 11 NA 9 NA 50 0.5 65 0.4 36 0.77 47H₂O11571 28 0.59 8 NA NO 23 NO1.62 9 NA 7 NA 8 NA N = 1 N = 2 15 NA 4 NA 9 NA 7 NA 14 NA 17 NA 2 NA S.epi P. acnes 3.5 μg/ml 50 μg/ml 20 μg/ml 8 μg/ml 3.5 μg/ml ScreeningExtract % Z′ % Z′ % Z′ % Z′ % Z′ Primary 47 6 NA 59 0.8 41 0.6 18 NA 320.6 N = 1 N = 2 NO  3.82 53 0 49 NA 25 NA 33 NA Secondary 47H11571 NO10.4 45 0.7 27 0.4 30 0.4 21 0.3 N = 1 N = 2 NO 11.4 57 0.5 41 0.6 310.4 26 0.3 47AC11571 2 NA 28 0.5 24 0.5 19 0.2 25 0.3 N = 1 N = 2 4 NA33 0.5 25 0.3 20 NA 22 NA 47CL11571 5 NA 54 0.7 38 0.3 42 0.7 22 0.6 N =1 N = 2 9 NA 60 0.5 53 0.7 47 0.4 28 0.3 47H₂O11571 15.5 5 NA 15 NA 15NA 19 0.3 N = 1 N = 2 3 NA 10 NA 13 NA 14 NA 23 0.1

Example 6. Improving Bioactivity for Anti-Acne Related ActivityAssociated with FD Extracts

The specific objective of these experiments was to optimize bioactivityof the extracts and identify the geographic localization of the mostpromising natural resource.

A collection program for FD was established for different geographicalregions on the west coast of Newfoundland and Labrador (Table 6).

TABLE 6 Extraction yields Extract Yield (g dry (g dry extract/ SamplesDate Collected Location weight) g dry plant) Fucus Oct. 6, 2014 Bradore,Quebec 7.29 10.40 Distichus #79 (BasinIsland) Fucus Oct. 9, 2013 Salmonpt. Bonne 4.35 6.21 Distichus #87 Bay, NF Fucus Oct. 10, 2013 Wild Cove,Bonne 6.19 8.78 Distichus #85 Bay, NF

Table 7 shows the anti-microbial activity of the three primary extractsobtained from different locations during the collection program inOctober 2013, prepared in accordance with the protocol presented inExample 1 (80% aqueous ethanol).

TABLE 7 Summary of anti-microbial activity of primary FD extracts #79,87 and 85 collected from different locations MRSA S. epi Mass 50 μg/ml20 μg/ml 8 μg/ml 50 μg/ml 20 μg/ml Extract (mg) % Z, % Z′ % Z′ % Z′ % Z′FD79 20 mg/ml 72.66 0.662 12.86 NA 7.968 NA 35.2 NA 18 NA N = 1 N = 269.96 0.724 11.24 NA 2.494 NA 61.34 0.124 9.13 NA FD87 20 mg/ml 97.380.926 76.01 0.539 10.31 NA 98.3 0.912 13.74 NA N = 1 N = 2 98.69 0.85974.47 0.702 5.164 NA 98.15 0.649 30.13 NA FD85 20 mg/ml 98.91 0.93 95.670.889 22.47 NA 98.99 0.912 71.49 0.505 N = 1 N = 2 98.87 0.862 92.470.645 18.93 NA 98.8 0.661 83.79 0.29 S. epi P. acnes Mass 8 μg/ml 50μg/ml 20 μg/ml 8 μg/ml Extract (mg) % Z′ % Z′ % Z′ % Z′ FD79 20 mg/ml2.467 NA NO 2.89  NO 2.329 4.92 0 N = 1 N = 2 7.951 NA 5.856 NA 6.3230.099 14.3 0 FD87 20 mg/ml 5.33 NA 7.896 NA NO 3.646 NO 4.857 N = 1 N =2 5.157 NA 31.46 0.191 NO 4.046 NO 2.454 FD85 20 mg/ml 17.43 NA 11.43 NANO 2.43 NO 6.231 N = 1 N = 2 NO 37.95 28.92 NA 1.35  NA 8.051 NA

This was followed by investigating different extraction conditions topotentially optimise bioactivity of FD extract #87. Three differentsolvent ratios were used (ethanol/water at 80, 50, and 25%). Extractionwas performed at 3 different temperatures (30, 50, and 80° C.) for eachsample (see FIG. 5).

TABLE 8 Extraction conditions for secondary fractionation of FD primaryextract #87 Yield (g dry extract/g Samples and Solvent ratio Extract drydry extract ID (ethanol:water) Temperature weight (g) seaweed) FD 87 A1A 1) 80° C. 0.49 10.0 A2 80% 2) 50° C. 0.44 8.0 A3 3) 30° C. 0.19 4.0 B1B 1) 80° C. 0.49 10.0 B2 50% 2) 50° C. 0.61 12.0 B3 3) 30° C. 0.61 12.0C1 C 1) 80° C. 1.14 22.0 C2 25% 2) 50° C. 0.64 12.0 C3 3) 30° C. 0.4510.0

Extraction yields were calculated for each extract. Yields ranged from4.0 to 22.0, when expressed as g of dry extract per g of dry seaweed(Table 8). It was noticed that using 25% aqueous ethanol at 80° C.increased the yield.

Table 9 shows that all secondary fractions showed high anti-microbialactivity (i.e. >75% inhibition) against MRSA, and S. epidermis, at aconcentration of 50 μg/ml. However, the activity against P. acnes showedinconsistent results that may be due to contamination of the originalextract or contamination of the bacterial culture.

To ascertain activity of FD extract on P. acnes, five (5) extracts wereselected from Table 9 and were further evaluated for their anti-P-acnesactivity at the following concentrations: 200; 100; 50; 25; 8 and 3.2μg/ml. Table 10 shows that primary or secondary FD extracts possessanti-P. acnes activity, albeit at concentrations of 200 and 100 μg/ml.

TABLE 9 Summary of anti-microbial activity of secondary extracts from FDprimary extract # 87 MRSA S. epi Mass 50 μg/ml 20 μg/ml 8 μg/ml 50 μg/ml20 μg/ml Extract (mg) % Z′ % Z′ % Z′ % Z′ % Z′ FD87B 20 mg/ml 88.820.561 79.12 0.847 33.25 0.372 92.39 0.794 25.1 0.026 N = 1 N = 2 89.140.863 82.86 0.836 26 0.677 98.72 0.908 13.27 NA FD87C 20 mg/ml 89.470.703 81.38 0.729 29.7 0.268 98.63 0.94  19.86 NA N = 1 N = 2 95.040.928 79.12 0.763 27.82 0.425 88.41 0.579 16.97 NA FD87A1 20 mg/ml 84.980.64 53.78 0.502 26.89 0.308 41.98 NA 14.08 NA N = 1 N = 2 79.61 0.63156.68 0.527 25.72 0.59  43.96 NA 0.244 NA FD87A2 20mg/ml 81.69 0.79255.52 0.457 38.55 0.58  60.05 0.75  7.798 NA N = 1 N = 2 86.12 0.90169.08 0.823 25.49 0.572 80.75 0.256 5.688 NA FD87A3 20 mg/ml 86.01 0.83862.36 0.522 30.68 0.446 34.04 NA 3.737 NA N = 1 N = 2 84.45 0.779 64.940.799 23.85 0.375 80.78 0.42  1.503 NA FD87B1 20 mg/ml 87.84 0.606 63.880.635 24.72 0.033 82.38 0.413 2.079 NA N = 1 N = 2 81.66 0.898 66.790.424 15.34 0.12  84.45 0.471 NO 5.946 FD87B2 20 mg/ml 88.69 0.828 79.990.694 27.69 0.104 80.04 0.755 11.94 NA N = 1 N = 2 84.06 0.646 74.520.604 20.69 NA 77.85 NA NO 6.064 FD87B3N 20 mg/ml 85.11 0.841 28.720.533 27.7 0.092 82.1 0.556 NO 386.8   N = 1 N = 2 84.35 0.648 72.140.789 17.48 NA 82.07 0.413 NO 6.428 FD87C1 20 mg/ml 88.99 0.784 70.680.741 21.32 NA 92.47 0.786 11.11 NA N = 1 N = 2 92.39 0.864 77.99 0.84515.85 NA 79.75 0.141 NO 5.149 FD87C2 20 mg/ml 90.35 0.774 78.08 0.69429.1 0.385 72.11 NA 10.9 NA N = 1 N = 2 91.21 0.757 68.65 0.199 14.610.214 93.14 0.62  6.218 NA FD87C3 20 mg/ml 95.26 0.853 73.53 0.382 23.74NA 86.87 0.645 10.21 NA N = 1 N = 2 90.64 0.618 64.71 NA 9.575 NA 45.67NA NO 12.03  S. epi P. acnes Mass 8 μg/ml 50 μg/ml 20 μg/ml 8 μg/mlExtract (mg) % Z′ % Z′ % Z′ % Z′ FD87B 20 mg/ml 7.787 NA NO 1.965 NO2.784 2.733 NA N = 1 N = 2 NO 6.343 NO 26.3   NO 9.488 NO 46.98  FD87C20 mg/ml 11.31  NA 7.354 NA 0.835 NA 11.28  0    N = 1 N = 2 NO 4.704 NO5.155 NO 3.819 NO 0    FD87A1 20 mg/ml 8.02  NA 0.931 NA NO 4.18  6.12 NA N = 1 N = 2 NO 7.937 NO 10.64  NO 29.44  NO 13.3   FD87A2 20 mg/ml3.418 NA 4.548 NA NO 3.557 1.959 NA N = 1 N = 2 0.337 NA 11.87  NA 8.152NA 6.886 NA FD87A3 20 mg/ml 0.826 NA NO 19.74  NO 4.399 1.838 NA N = 1 N= 2 2.948 NA NO 4.709 1.524 NA 1.17  NA FD87B1 20 mg/ml NO 9.984 NO3.674 NO 5.28  23    0.184 N = 1 N = 2 NO 3.499 NO 2.638 NO 2.84  NO3.253 FD87B2 20 mg/ml 0.261 NA 3.89  NA NO 10.88 7.544 NA N = 1 N = 2 NO3.711 0.146 NA NO 8.029 NO 4.023 FD87B3N 20 mg/ml NO 5.978 NO 6.528 NO4.566 1.065 0    N = 1 N = 2 NO 3.552 NO 3.252 NO 2.72  NO 0    FD87C120 mg/ml NO 9.48  1.17  NA NO 2.189 NO 4.701 N = 1 N = 2 NO 5.039 NO3.289 NO 2.357 NO 2.372 FD87C2 20 mg/ml 0.738 NA NO 93.36  NO 3.4584.705 NA N = 1 N = 2 NO 2.146 NO 3.09  NO 2.471 NO 2.383 FD87C3 20 mg/mlNO 16.29  5.218 NA NO 2.526 NO 4.192 N = 1 N = 2 NO 1.82  NO 2.564 NO2.293 NO 2.181

TABLE 10 Summary of anti-P. acnes activity of five selected extracts P.acnes P. acnes Mass 200 μg/ml 100 μg/ml 50 μg/ml 200 μg/ml 100 μg/ml 50μg/ml Extract (mg) % Z′ % Z′ % Z′ % Z′ % Z′ % Z′ FD78 20 mg/ml 25.120.305 NO 7.781 NO 2.285 NO 11.16  NO 5.583 4.053 NA N = 1 N = 2 37.170.469 6.959 NA  0.413 NA 2.461 NA NO 6.135 5.692 NA FD87 20 mg/ml 50.420.783 60.02 0.578 23.55 NA NO 6.515 NO 3.392 NO 16.21 N = 1 N = 2 48.470.621 64.37 0.559 26.32 0.054 0.605 NA NO 3.392 NO 10.89 FD87A2 20 mg/ml49.52 0.691 49.52 0.691  4.685 NA NO 771.1   NO 6.039 8.276 NA N = 1 N =2 46.46 0.511 55.4 0.64   7.84 NA 6.099 NA NO 674.5 9.196 NA FD87B2 20mg/ml 45.96 0.705 3.516 NA NO 2.54  NO 10.84  5.189 NA 12.43 NA N = 1 N= 2 46.25 0.498 10.35 NA NO 4.072 NO 7.747 2.513 NA 7.142 NA FD87C3 20mg/ml 53.41 0.865 47.71 0.46  18.99 NA NO 10.36  4.344 NA 17.62 NA N = 1N = 2 58.25 0.674 43.71 NA 14.77 NA NO 7.638 NO 2307 7.33 NA

Despite inconsistent results for P. acnes, it remains clear that Fucusdistichus, whether as a primary extract or as secondary fractions,possesses highly interesting anti-microbial activity against nosocomialinfections and acne.

Example 7: Evaluation of Fucus Distichus Activity Against StaphylococcusPseudintermedius

The objective of this project was to study the growth kinetic ofStaphylococcus pseudintermedius (ATCC® 49051™) and test the activitiesof two FD extracts: FD85 and FD87 (in 80% ethanol as defined in Example2).

Four samples (FD85 and FD87: in their original form, or FD85a and FD87a:previously pasteurized for 3 h) were assayed on antimicrobial screeningat 10³ CFU/mL (see FIG. 6) in Mueller Hinton broth. The antibacterialactivity was measured after 24 hours of incubation (see FIG. 7) withluminescence technology and is shown in Table 11 and FIG. 8.

TABLE 11 Antimicrobial activity of FD extracts against SP (ATCC ®49051 ™) Mueller Hinton mass 50 μg/ml 20 μg/ml 8.0 μg/ml 3.2 μg/mlExtract (mg) % Z′ % Z′ % Z′ % Z′ FD85a N = 1 20 mg/ml 98.34 0.308 27.170.105 16.64 NA 13.9 NA (Pasteurized) N = 2 98.81 0.889 12.9 NA 9.993 NA11.82 NA FD85 N = 1 17.55 mg/ml   96.83 0.87 33.98 NA 15.76 NA 12.44 0  N = 2 89.49 0.347 25.77 NA 14.61 NA 9.03 NA FD87a N = 1 20 mg/ml 98.320.917 40.54 NA 4.977 NA 8.776 NA (Pasteurized) N = 2 98.65 0.872 64.36NA 8.041 NA NO 99.41 FD87 N = 1 13.65 mg/ml   99.12 0.326 87.29 NA 13.13NA 12.22 NA N = 2 99.26 0.894 97.33 0.84  7.251 NA 7.352 NA Legend: NO =No inhibition, grey = INH25-50%, italics = INH 50-74%, and bold = INH75-100% (a strong antimicrobial response)

Based on the results obtained, extracts FD85 and FD87 whether untreatedor previously pasteurized (extracts “a”) show high antimicrobialactivity against Staphylococcus Pseudintermedius at 50 μg/mL.

Example 8: Clinical Trial of Fucus Distichus Extracts AgainstStaphylococcus Pseudintermedius in Dogs

Protocol Title: An open label study of Brown Seaweed (FD) Extract as aTopical Staph Infection Therapy for in dogs.

Purpose

Staph infections are common disease of dogs (mainly S.pseudointermedius). This study will investigate whether treatment with atopical gel (or drops) containing FD Seaweed extract is an effectivetherapy in twenty (20) otherwise healthy dogs with Staph Infections. Thehypothesis is that treatment with a topical formulation containing FDextract will result in a significant improvement in Staph Infectionsafter 14 days of treatment.

Primary Outcome Measures

-   -   Negative Culture for Staph Infection after 14 days of treatment        (or less) with FD Seaweed extract.

Materials Under Test/Product Formulation

The topical preparation will consist of: 5 mg of FD seaweed extract per100 mL (50 μg/mL) of usual gel or liquid formulation excipients (5% w/vof the total formulation).

Study Flow/Treatment Plan Visit 1

-   -   Clinic Visit and Vet Assessment    -   Medical History    -   If deemed by Vet the dog as a Staph Infection then:        -   A Culture will be performed to determine the type of Staph            Infection        -   The Infected Area will be prepped for Treatment        -   A Topical Gel Application (One Daily) for 14 days or as            otherwise directed by the vet of FD seaweed extract will be            applied to the affected areas.            -   If the infection is in the ear—Three (3) drops of FD                seaweed extract will be used.

Visit 2 (14 Days or Prior as Deemed by Vet)

-   -   Vet Assessment    -   A Culture will be performed to determine if the of Staph        Infection has cleared    -   Adverse Event Query

Visit 3 (1-2 Weeks Post Treatment)

-   -   Vet assessment to determine that the infection has not returned.

1. A method for treating a Propionibacterium acnes bacterial infectionin a mammal, said method comprising administering to said mammal agrowth-inhibiting concentration of a composition comprising a solventextract from a Fucus distichus (FD) seaweed.
 2. The method of claim 1,wherein said mammal is a human.
 3. The method of claim 1, wherein saidsolvent is aqueous ethanol.
 4. The method of claim 3, wherein saidsolvent is between 25% and 80% aqueous ethanol.
 5. The method of claim1, wherein said seaweed is previously freeze-dried, grinded into powderform and defatted prior to solvent extraction.
 6. The method of claim 5,wherein said powder is defatted with hexane.
 7. The method of claim 1,said extract being further fractionated with a solvent selected from thegroup consisting of organic and inorganic solvents.
 8. The method ofclaim 7, wherein said fractionation solvent is selected from the groupconsisting of: hexane, ethyl acetate, chloroform, water, and mixturesthereof.
 9. The method of claim 1, wherein said extract is an 80%ethanol extract of said FD or a: hexane; ethyl acetate; chloroform; orwater fraction from said 80% ethanol extract.
 10. The method of claim 1,wherein said extract is in dried form or in solution.